SUMMARY The goal of this renewal application is to define mechanisms that drive the assembly and organization of enterocyte microvilli: actin bundle-supported membrane protrusions that extend from the apical surface into the gut lumen and play essential roles in nutrient absorption and barrier function. Numerous intestinal diseases are linked to the destruction or malformation of microvilli, underscoring the critical physiological importance of these protrusions. The surface of a single mature enterocyte is populated by hundreds of microvilli, which form a tightly packed array known as the ?brush border?. Tight packing maximizes the number of microvilli, the apical holding capacity for membrane-associated nutrient processing and host defense factors, and thus the functional capacity of the cell. Although mechanisms of brush border assembly remain poorly understood, our laboratory has begun to make significant progress on this problem. Groundbreaking studies performed during the initial funding period led to the discovery of a fundamental mechanism that enterocytes use to organize and optimize the packing density of microvilli. Briefly, we discovered two brush border-specific protocadherins, CDHR2 and CDHR5, which form adhesion complexes that physically link the tips of adjacent microvilli on mature villus enterocytes. In cultured cells lacking these factors, brush border assembly is impaired such that microvilli are disheveled with significantly reduced packing density. We also identified factors that interact with the cytoplasmic tails of both protocadherins, including the scaffolds USH1C and ANKS4B, and the actin-based motor, MYO7B. We refer to the entire five-protein complex as the intermicrovillar adhesion complex (IMAC). Our published work suggests that USH1C/ANKS4B/MYO7B form a transport module that delivers CDHR2- dependent adhesion links to the distal tips of microvilli. Preliminary results also show that adhesion links form between microvilli on the surface of immature cells in the crypt, although they do not yet target to microvillar tips, suggesting that IMAC transport is activated later, perhaps during the crypt-villus transition. Finally, newly developed CDHR2 KO mice exhibit striking defects in brush border morphology that have consequences for enterocyte differentiation and function. In light of these findings, we propose our central hypothesis: USH1C induces clustering of MYO7B motors, which activates transport of CDHR2-dependent adhesion links to microvillar tips and drives functional maturation of the brush border as cells exit the crypt. To test this model, we will use state-of-the-art super-resolution microscopy, new forms of electron microscopy, structural biology, and assays of epithelial function to: (Aim 1) determine how IMAC formation and localization at microvillar tips contribute to BB assembly and enterocyte function in vivo, (Aim 2) dissect mechanisms that regulate MYO7B- driven IMAC transport to microvillar tips, and (Aim 3) define the structural basis of CDHR2-dependent adhesion. Completion of these studies will lead to new insight on mechanisms driving brush border assembly, the basis of diseases characterized by brush border perturbation, and the morphogenesis of transporting epithelial cells.